Global genome initiatives including the Human Genome Project have generated enormous amounts of information, spawned new technologies and catalyzed the emergence of a new type of biology which attempts to build biological knowledge from the global analysis of biological systems and pathways from genes to proteins. Consequently, there is a growing need to develop means of global assessment that will ultimately provide a more clear understanding of biology at the functional protein level. Because both protein activity and turnover are tied closely to protein post-translational modification (e.g., phosphorylation and ubiquitination), new technologies must be developed to allow for the large-scale identification, characterization, and quantification of protein post-translational modifications. Reversible protein phosphorylation is a general event affecting countless cellular processes. In addition to a critical role in normal physiology, malfunctions in protein phosphorylation have been implicated in the etiology of many diseases such as diabetes, cancer, and Alzheimer's disease. Although protein phosphorylation is an intense research area, the large-scale isolation and identification of phosphorylation sites is an unsolved problem. We propose to develop a series of novel technologies for the isolation, identification, and quantification of proteins modified by phosphorylation on a large (ultimately global) scale in biological samples from cells and tissues. This new strategy will be a highly integrated approach using strong cation exchange (SCX) chromatography for phosphopeptide enrichment, nano-scale microcapillary liquid chromatography tandem mass spectrometry (LC-MS/MS) for phosphopeptide sequencing, and stable isotope incorporation for quantitative phosphoproteomics. In addition, we will apply the strategy to identify tens of thousands of phosphorylation sites from both primary tissues and cell lines.